This invention relates to a non-reducing end modified oligosaccharide derivative useful as a substrate for measuring .alpha.-amylase activity, a process for producing the same, and .alpha.-amylase activity assay using said derivative as a substrate.
Measurement of .alpha.-amylase activity in samples, particularly in saliva, pancreatic juice, blood and urea in human living bodies is important in medical diagnosis. For example, .alpha.-amylase activity in blood and urea remarkably increases compared with a normal value when suffered from pancreatitis, cancer of the pancreas, and parotid gland. As to processes for measuring .alpha.-amylase activity, various processes have been reported. These processes can be divided into two groups, one of which uses long-chain natural products such as starch, amylose, amylopectin, etc. and modified materials thereof, and another of which uses oligosaccharides having 3 to 7 glucose residues.
Among these processes, there is widely used a coupling enzyme process using as a substrate maltooligosaccharide derivatives having a uniform structure and a clear substrate, that is, having a color forming group at the reducing end and having a modified group at the 6-position or 4- and 6-positions of the non-reducing end glucose unit. Since these substrates do not become a substrate for a coupling enzyme such as .beta.-glucosidase, .beta.-glucosidase, glucoamylase, etc. and the detection can be carried out by measuring the amount of colored substance released, they are particularly significant as substrates for measuring .alpha.-amylase activity. Particularly, maltooligosaccharide derivatives modified only at the 6-position of non-reducing end glucose and disclosed in Japanese Patent Unexamined Publication Nos. 63-170393 (EP 0252525) and 61-83196 (U.S. Pat. No. 4,762,917) are highly reactive as a substrate for .alpha.-amylase and excellent in storage stability, so that they are substrates for measuring .alpha.-amylase activity with almost high completeness. But they have a problem in that the conversion at the synthesis of these maltooligosaccharide derivatives is so low that they are not suitable for practical production.
Therefore, novel non-reducing end modified oligosaccharide derivatives capable of becoming excellent substrates for measuring o-amylase and able to be synthesized in a high yield are now desired.